HIV Drotease (HIV PR) is an important drug target due to its crucial role in the maturation of HIV virions. The emergence of drug-resistant strains of the virus has prompted increased study into the nature of the resistance, with the goal of designing more effective drugs. Examination of public and private sequence databases of drug experienced HIV strains may yield proteases with the unique resistance mechanism of altered substrate specificity. These proteases will then be cloned, expressed and biophysically characterized with respect to their kinetic parameters, substrate specificity and inhibitor profiles. This will provide a molecular image of what the enzyme recognizes and help identify the determinants of resistance. Reversion mutagenesis will then be used to evaluate the contribution of specific amino acids to resistance. Selected variants will be pursued at a structural level using both X-ray crystallography and NMR spectroscopy. This will provide an atomic level understanding of the role of specific amino acids to altered conformational flexibility, a key mechanism of resistance. Significant progress has been made on variants of HIV PR that exhibit altered substrate specificity and are resistant to current protease inhibitors. The results of these studies will enable us to understand the mechanism by which HIV PR becomes resistant to small-molecule inhibitors. This, in turn, may lead to the design of more effective anti-proteolytic drugs that continue to reduce viremia in HIV-positive individuals. In a closely related project we will study the serine protease encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) as a potential target for KS, the most common neoplasm of patients with AIDS. Demonstrating the role of KSHV protease in cell culture has proven difficult because of a lack of potent, specific inhibitors. We will determine the basis of substrate recognition of KSHV PR. This information will assist us in developing a selective chemical probe targeting the active site of the protease. We will also target the interface of this unique dimeric serine protease with dimerization disrupters. Our efforts will be guided by X-ray and NMR structural analysis on the dimeric and monomeric forms of the protease where we have already made significant progress. Herpesvirus protease inhibition will reveal the importance of protease activity on viral replication. The reagents obtained should be easily adaptable to all of the herpesviruses, including the herpes simplex viruses (HHV1 & HHV-2), Varicella-Zoster virus (HHV-3), Epstein-Barr virus (HHV-4), and human cytomegalovirus (HHV-5).